Dynamic imaging of protein machinery for synaptic plasticity in neuronal dendritic spines
Lead Research Organisation:
University of Bristol
Department Name: Biochemistry
Abstract
Long-term synaptic plasticity underlies learning and memory and the tuning of neural circuitry. A central process involved in plasticity of excitatory synapses is dynamic changes in the size and shape of dendritic spines. These dendritic protrusions compartmentalise postsynaptic proteins, and concentrate biochemical signals. Spines shrink following the induction of long-term depression (LTD), and grow during long-term potentiation (LTP). In addition, aberrant spine morphology is a critical factor in brain disorders such as schizophrenia, autism spectrum disorders and stroke. Dendritic spine structural plasticity involves a complex network of signalling pathways converging on protein complexes that regulate the actin cytoskeleton. The timing and magnitude of protein interactions following stimulation and how these variables influence changes in spine morphology are unclear.
Protein interactions can be analysed in live cells using FLIM-FRET (Fluorescence Lifetime Imaging - Förster Resonance Energy Transfer), which provides a dynamic measure of the proximity of two fluorophores, and hence of two proteins with appropriate fluorescent tags.
The aim of this project is to analyse relevant protein-protein interactions in dendritic spines using FLIM-FRET in response to stimuli that induce plasticity. The study will focus on PICK1, which inhibits actin polymerisation and is essential for LTD. PICK1 interacts with a number of proteins that are crucial for plasticity, including Arp2/3, small GTPases, PKC, and calcineurin. FRET pairs will be generated to analyse interactions between PICK1 and each of these proteins in turn. Following initial characterisation to determine the feasibility of each FRET pair, FLIM will be used to measure changes in fluorescence lifetime as a readout for FRET, and hence a measure of protein interaction. This will be done in real time following NMDA stimulation to induce LTD. Spine size will be recorded simultaneously by analysing an appropriate unconjugated fluorescent protein as a morphological marker.
Our hypothesis is that the extent of these specific protein interactions governs the degree of spine shrinkage. Computational methods will be employed to test this hypothesis and build a model of NMDA-induced spine shrinkage based on specific protein-protein interactions. The acquired imaging data will be combined with previous knowledge about cytoskeletal dynamics to develop a mathematical model taking into account other molecular mechanisms that govern spine size during LTD. This will be extended to reconstruct a stochastic computational model of the system's dynamics to estimate the effect of stochasticity, and hence assess the efficiency and fidelity of changes in spine size.
Protein interactions can be analysed in live cells using FLIM-FRET (Fluorescence Lifetime Imaging - Förster Resonance Energy Transfer), which provides a dynamic measure of the proximity of two fluorophores, and hence of two proteins with appropriate fluorescent tags.
The aim of this project is to analyse relevant protein-protein interactions in dendritic spines using FLIM-FRET in response to stimuli that induce plasticity. The study will focus on PICK1, which inhibits actin polymerisation and is essential for LTD. PICK1 interacts with a number of proteins that are crucial for plasticity, including Arp2/3, small GTPases, PKC, and calcineurin. FRET pairs will be generated to analyse interactions between PICK1 and each of these proteins in turn. Following initial characterisation to determine the feasibility of each FRET pair, FLIM will be used to measure changes in fluorescence lifetime as a readout for FRET, and hence a measure of protein interaction. This will be done in real time following NMDA stimulation to induce LTD. Spine size will be recorded simultaneously by analysing an appropriate unconjugated fluorescent protein as a morphological marker.
Our hypothesis is that the extent of these specific protein interactions governs the degree of spine shrinkage. Computational methods will be employed to test this hypothesis and build a model of NMDA-induced spine shrinkage based on specific protein-protein interactions. The acquired imaging data will be combined with previous knowledge about cytoskeletal dynamics to develop a mathematical model taking into account other molecular mechanisms that govern spine size during LTD. This will be extended to reconstruct a stochastic computational model of the system's dynamics to estimate the effect of stochasticity, and hence assess the efficiency and fidelity of changes in spine size.
Organisations
People |
ORCID iD |
| Jonathan Hanley (Primary Supervisor) | |
| Georgiana Stan (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M009122/1 | 01/10/2015 | 31/03/2024 | |||
| 1788559 | Studentship | BB/M009122/1 | 01/10/2016 | 31/03/2021 | Georgiana Stan |
| Description | The main focus of my project has changed a lot from when I first started. I was hopeful that I could use FLIM-FRET, a powerful imaging tool used to investigate protein-protein interactions, in order to look at the interactions of a specific protein, namely PICK1, in live neurons which had been treated with chemicals to induce synaptic plasticity, the cellular process behind memory formation. After months of trial I realised that there are too many limitations to be able to image live stimulated neurons and resolved to work with fixed samples instead. This is how I obtained my first result which indicated that PICK1 is more self-associated during synaptic plasticity. My next challenge involved finding a reliable way to look at dimers/oligomers in order to decipher the regulatory mechanisms behind PICK1 self-association. Finally, I was able to show that calcium concentration affects PICK1 dimerisation, and that this effect is probably mediated by acidic regions within the BAR domain and N-terminus of the protein. I am currently working on investigating how other factors influence PICK1 dimerisation, as well as planning to conduct my experiments using neuronal samples in order to understand the importance of the PICK1 dimer for synaptic plasticity. |
| Exploitation Route | I believe my research is important because there is little information available about PICK1 dimerisation. Considering that the majority of proteins from the same family as PICK1 form functional dimers, it would be interesting to see how PICK1 compares to these in terms of regulatory mechanisms controlling self-association. If dimerisation and further oligomerisation is a strict requirement for the function of PICK1, it opens up new avenues for therapeutic intervention in diseases such as neurodegenerative disorders, addiction and schizophrenia. I also find value in my research as a way of getting closer to deciphering how memory works and believe my work could be continued by others in this direction. |
| Sectors | Pharmaceuticals and Medical Biotechnology |